Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 9
Issue 4
Available Online: http://www.ejpau.media.pl/volume9/issue4/art-50.html


Aleksander Gonkiewicz, Kazimierz Nosal
Department of Pomology, University of Agriculture in Cracow, Poland



The aim of auxin application in thinning of flowers and young fruits is to reduce the number of set fruits and to obtain a high quality fruit yield. Six-year old plum trees ‘Stanley’ and ‘Iroquois’ cultivars treated with a solution of potassium salt of alpha-naphtaleneacetic acid (NAA) after blooming were used in the experiment. The applied concentrations varied from 30 to 160 mg NAA l-1. Microscopic preparations of flowers and young fruits were made using fluorescence and paraffin methods. There was noted a slight stimulating effect of NAA treatment on the fertilization process. It was also found that the applied auxin induced callose saturation and clogging up of conductive bundles and the chalazal part of the ovule. The inhibition of assimilate translocation to the ovule due to the clogging up of the chalazal part brought about an inhibition in endosperm development and resulted in embryo degeneration.

Key words: callose, NAA, plum, thinning.

Abbreviations: NAA – potassium salt of alpha-naphtaleneacetic acid


Many plum cultivars show a tendency towards excessive fruit setting, which resulting in high yields of very small fruit of low commercial value. The excessive fruit setting also inhibits the formation of flower primordia for the next year bringing about the phenomenon of biennial bearing. It can be prevented by the thinning of flowers and young fruit conducted in the years of intensive fruit setting. For this aim trees are sprayed with various preparations. Almost all available plant hormons and syntetic growth regulations were tested, but NAA has been used the most often during 40 last year [19]. Auxin NAA is especially useful to plum thinning [8], but the role of NAA in mechanism of fruit dropping is not quite clear yet [21]. Several theories concerning the mechanism of NAA effect have been described in the literature. One of them is based on the action of ethylene whose synthesis is stimulated by exogenous auxin [4]. The influence of NAA on intensity of fruit drop mechanism is very changeable and really difficult to predict [19]. The aim of this research was to specify the mechanism of fruit dropping after thinning using potassium salt of alpha-naphtaleneacetic acid.


Research was carried out in 1999-2001, on six-year old ‘Stanley’ and ‘Iroquois’ plum trees (Prunus domestica L.). The spraying with a solution of potassium salt of alpha-naphthaleneacetic acid (NAA, C12H9O2K, Sigma) was conducted by the end of the blooming period, however, the main indication of the time of treatment was a distinctly visible abscission layer between the perianth and the pedicle (phot. 1), signaling the fertilization [11]. In the first year of the experiment with the cultivar ‘Stanley’ concentrations of NAA were 30 and 40 mg l-1 and in the successive years 30, 40 and 50 mg l-1. In the second year the cultivar ‘Iroquois’ treated with high concentrations of 40, 80 and 160 mg NAA l-1 was included in the experiment. Control trees were not treated.

Phot. 1. Abscission layer (al) between the perianth and the pedicle

In the investigation of the effect of NAA treatments on the anatomical development of young fruits, microscopic observations of samples prepared using fluorescence microscopy and paraffin methods were carried out. The examined flowers and young fruits of the cultivars ‘Stanley’ and ‘Iroquois’ were sampled from a control plot (untreated) and from plots with trees treated with NAA. The observations were conducted during about two weeks from the time of full blooming, however, the presented results concern four days, beginning from the second day after the auxin treatment.

Fluorescence method. The aim of microscopic examination using the fluorescence method was to determine the consequential effect of NAA on fertilization percentages and the accumulation of callose in the vascular tissue of young fruits. Specimens were fixed with FAA [20] for 24 hours, then were soaked in NaOH 30% for 24 hours and next (without a rinse) in H2O2 6% at 36°C for 1 hour (to bleach) and staining in aniline blue (100 ml distillated water, 2.6 g potassium phosphate tribasic pure, 100 mg aniline blue; pH = 12.3; time of staining about 20 min. in underpressure pump). Smear specimens were examined at fluorescence microscope (Russian microscope МЛ-2, filter уфс 6-5, 310-380 nm) with UV rays according to Martin [12] in modification Lech [10]. Callosa is bright yellow if observed using this method. In one replication were young fruits tested from one day. The statistic calculation consisted results from four days, from second to sixth day after treatment. The growing pollen tubes into micropyle signified a fertilization (phot. 2, 3, 4) This methodology is based on high correlation between fruit setting and fertilization assessing in this way [10]. Data were analyzed using Stat program for Windows with t-Duncan test at α = 0.05.

Phot. 2. Fertilization – a pollen tube (pt) growing into the nucellus. 11 days after blooming. Smear preparation in fluorescence microscope. Control

Phot. 3-4. Pollen tube (pt) growing throught the micropyle and reaching the nucellar tip (nt);
ii – inner integuments. Smear preparation in fluorescence microscope (3), paraffin preparation (4). Control

Paraffin method. The aim of the investigation of microscopic preparations using the paraffin method was to determine the effect of NAA on the anatomical changes in plum fruits. For paraffin preparations of young fruits samples were taken every day beginning from the day before the NAA application up to the 8th day after the treatment. Six young fruit (fruitlets) were prepared from combination every day. Specimens were fixed with Carnoy liquid and saturated with paraffin wax (54-56°C melting point). After slicing (5-7 µm of thickness) they were stained with Heidenhain haematoxylin [6] and observed in microscope (Hund Wetzlar H500).


It was found that percentage of fertilized flowers (phot. 2) is exceeded in auxin treatments than in the control (tab. 1). These results suggested that stimulating effect of NAA on the fertilization is connected with ethylene production and characteristic of this gas [15, 16].

Table 1. Percentage of fertilized ovules of the cultivars ‘Stanley’ and ‘Iroquois’ as depending on the applied NAA concentration in 1999-2001







25.0 a*

25.0 a

32.5 a

30 mg NAA l-1

25.0 a

33.3 b

36.7 ab

40 mg NAA l-1

27.8 a

21.1 a

33.8 a

50 mg NAA l-1


34.4 b

47.5 b



35.8 a

33.5 a

40 mg NAA l-1

34.5 a

39.2 a

80 mg NAA l-1

41.6 ab

55.8 b

160 mg NAA l-1

47.8 b

51.2 b

*Values marked with the same letter do not differ significantly at α = 0.05.
**No investigation

Microscopic studies using the fluorescence method showed an increased percentage of flowers with the chalazal part and vascular bundles of the ovule with callose after the auxin treatment (tab. 2). Vascular bundles are connected with the chalazal part of ovule (phot. 5). Bundles transport nutritive compounds to the nucellus and make possible its correct development (phot. 6-7). Callose deposited all chalazal part (phot. 8-9) and vascular bundles caused block assimilate flow and preventing the normal development of ovule.

Table 2. Percentage of ovules in cultivars ‘Stanley’ and ‘Iroquois’ where callose was found in the chalazal part as depending on the applied NAA concentration in 1999-2001







9.5 a*

5.8 a

7.9 a

30 mg K-NAA l-1

9.2 a

5.8 a

7.3 a

40 mg K-NAA l-1

18.3 b

4.4 a

5.3 a

50 mg K-NAA l-1


20.0 b

5.9 a



18.3 a

14.2 a

40 mg K-NAA l-1

20.0 a

26.6 b

80 mg K-NAA l-1

66.5 b

43.3 c

160 mg K-NAA l-1

78.7 c

71.2 d

*Values marked with the same letter do not differ significantly at α = 0.05.
**No investigation

Phot. 5. Longitudinal section of young fruit (fruitlet) cv. Stanley: vascular bundle (vb), chalazal part (chp), ovule (ov). Eight days after blooming. Paraffin preparation. Control

Phot. 6-7. Nucellus without callose in chalazal part. Cultivar Stanley (6) and Iroquois (7). 13 days after blooming. Smear preparations in fluorescence microscope. Control

Phot. 8-9. Luminous callose in chalazal part of nucellus. Cultivar Iroquios. 13 days after full bloom, 3 days after 160 mg NAA l-1. Smear preparations in fluorescence microscope

The obtained data show that the blocking of the chalazal part of ovule with callose brings about the fruit drop (tab. 3). This finding agrees with data reported by Law and Yeung [9], who observed that the presence of callose in cell walls of the ovule is a symptom of its future degeneration. Mirza [13] observed a close connection between the development of seeds in dropped bean flowers and the deposition of callose in vascular bundles. The results of the presented experiment suggest that the chalazal part with callose blocks the inflow of assimilates bringing about the degeneration of the ovule and hence the premature fruit drop. Different authors also confirm these results. Dittmann and Stosser [5] found the presence of callose in the chalazal part of ovule of prematurely dropping fruits while Rodrigo and Herrero [18] and Pimienta and Polito [14] observed dying back ovules with callose in their chalazal part. These authors report that the deposition of callose within chalaza blocks the assimilate flow to the ovule, bringing about its degeneration. They also showed the decreasing content of carbohydrates in ovules after the chalazal part was blocked. However, it is still unclear if the synthesis of callose and deposition in chalazal part are the cause of the ovule degeneration or its result. According to Arbeloa and Herrero [2] the synthesis of callose precedes ovule degeneration. This agrees with the results of researches showing the presence of callose two days before distinct histological signs of the ovule degeneration [14]. The results of experiments by Aloni et al. [1] suggest that this auxin is not the direct factor inducing the synthesis of callose. They observed that exogenous NAA induced the removal of callose from the sieve tissue of shoots. If NAA does not directly influence the synthesis of callose, ethylene could be the responsible factor.

Table 3. Percentage of fruit setting of ‘Stanley’ and ‘Iroquois’ depending on the applied NAA concentration in 1999-2001







25.2 b*

15.6 b

21.4 a

30 mg NAA l-1

22.9 ab

11.1 a

19.8 a

40 mg NAA l-1

18.9 a

12.3 ab

21.6 a

50 mg NAA l-1


12.4 ab

19.0 a



24.1 c

12.0 b

40 mg NAA l-1

9.3 b

7.2 ab

80 mg NAA l-1

5.1 ab

2.9 a

160 mg NAA l-1

0.6 a

0.5 a

*Values marked with the same letter do not differ significantly at α = 0.05.
**No investigation

The inhibition of pro-embryo development also resulted from the inhibited growth of the ovule. The structure of embryo sac and endosperm was correctly developed in control (phot. 10). As first effects of exogenous auxin application, inhibition of endosperm development was observed (phot. 11). Next effects were observed on the second day after treatment. The inhibition of pro-embryo development was visible (phot. 12). Complete deformation of pro-embryo and the inhibition of endosperm development are visible on the third day after the NAA treatment (phot. 13). Additionally, granulation of endosperm was observed. Endosperm was lumpy and stuck together around wall of embryo sac.

Phot. 10. Normally developing pro-embryo (pe) and endosperm (en). Control. 10 days after blooming. Paraffin preparation

Phot. 11. Fragment of the embryo-sac. Inhibited development of endosperm (en), cv. Iroquois, 11 days after blooming and 1 day after 160 mg NAA l-1 treatment. Paraffin preparation

Phot. 12. Fragment of embryo-sac. No endosperm development (en) and the beginning of pro-embryo deformation (pe), cv. Iroquois, 12 days after blooming, 2 days after 160 mg NAA l-1 treatment. Paraffin preparation

Phot. 13. Embryo-sac. Pro-embryo deformations (pe), inhibited development and granulation of endosperm (en), cv. Iroquois, 14 days after blooming, 4 days after 160 mg NAA l-1 treatment. Paraffin preparation

Pro-embryo deformation could have resulted from endosperm inhibition [17]. Vitagliano et al. [22] reported similar results. They applied a solution of an NAA derivative and ethephone to peach trees. In microscopic preparations they found deformation of the ovule after the application of an NAA solution and the stunted growth and destruction of embryos after an ethephone treatment. These authors also found a rapid increase in NAA-induced ethylene. Byers et al. [3] reported a similarly destructive effect of synthetic auxin (2,4,5-TP). They recorded reduced numbers of seeds and a decrease in apple size. The results quoted above and these obtained in the presented experiment suggest that the destructive changes in the ovule are due to an increased ethylene concentration [7], however, it is difficult to state whether they are an intermediate or direct effect of its appearance. It is also difficult to state whether ethylene stimulates the production of callose, which blocks the chalazal part and brings about degeneration within the embryo sac, or directly affects the inhibition of endosperm and pro-embryo development, thus resulting in the synthesis of callose. In the presented experiment both types of symptoms were noted almost at the same time, that is 3-4 days after the NAA treatment.


The research was supported by the State Committee for Scientific Research (KBN) grant No. G-1360/KS/01-02.


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Accepted for print: 13.12.2006

Aleksander Gonkiewicz
Department of Pomology,
University of Agriculture in Cracow, Poland
29 Listopada 54, 31-425 Cracow, Poland
email: agonkiewicz@ar.krakow.pl

Kazimierz Nosal
Department of Pomology,
University of Agriculture in Cracow, Poland
29 Listopada 54, 31-425 Cracow, Poland

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